Sequencing the blood brain barrier in AD

Gabriel Mateus Bernardo Harrington

Research Associate | Webber Lab

2023-09-18

Glossery

  • Infarction: tissue death due to inadequate blood supply
    • Result of prolonged ischaemia, typically due to an artery blockage, rupture or vasoconstriction
    • Resulting lesion is refereed to as an infarct
  • Ischaemia: insufficient supply of oxygen/nutrition to tissue due to disrupted blood supply
  • Small Vessel Disease (SVD): what it sounds like, narrowing blood vessel, can cause ischaemia, increase stroke risk, etc
  • Smooth Muscle Cells (SMC)

Vascular dementia - epidemiology

  • Second most common dementia (~15-30%), and most common secondary dementia
  • Post-stroke 15-30% subjects develop dementia (Pendlebury and Rothwell 2009)
    • Some argue should be viewed as a subtype due to differing pathophysiology

Image depicting 4 MRI brain scans with various vascular damage

Structure of the neurovasculature

An image depicting the cellular structure of the blood vessels in the brain.

Vascular segmentation

An image depicting the cellular segmentation of a blood vessel in the brain

Blood Brain Barrier project - study design

  • 20 cases + 20 controls post-mortem human brain tissue
  • Parenchyma and vascular fractions (80 samples total) > single cell seq (10X)
  • Genotyping pending

A cartoon deliniating the parenchyma and vascular components of the brain

Data processing

  • Using scFlow
  • Standardised single cell seq pipeline written in Nextflow by Nathan Skene Lab
  • Aspirational goal for informatic pipelines in general for DRI
  • Pseudobulk to be added (by me probably, so Gods help us all)
  • It’s neat though, promise
  • Data will be on ADDI platform, with code and pipeline available for use

PCA

Two PCA plots with the first two PCs, coloured by sample diagnosis and sample type (parenchyma/vascular). The diagnosis does not seperate well along either PC, but the sample type seperates well across PC 1.

UMAP

UMAP plot coloured by diagnosis. Samples are relatively evenly distributed through the clusters

UMAP plot coloured by sample type. Samples are clearly seperated with a large cluster of vascular cells on the right and the parenchymal cells on the left.

UMAP 2 - electric boogaloo

UMAP plot coloured by cell type. There is sepration of the vascular cell types in the same cluster as the prior plot and the neuronal cells in the other clusters.

UMAP plot coloured by batch. There is a mixture of cells from each batch across all clusters, indicating the batch integration is working correctly

UMAP 3 - the empire strikes back?

Manual annotations based on Yang et al. (2022)

They have a neat shiny app for browsing their data here

UMAP plot coloured by manual cell type annotations. Similarly to the prior UMAP plots, the vascular cell types are in the large cluster to the right, and the neuronal cells are in the other clusters.

Pseudobulk

Volcano plots per celltype of DESeq2 pseudobulk results. The are no significant genes for the ambiguous, fibroblast and T-cells, the all other celltypes have significant hits.

Full pseudobulk gene list

Initial Pathways

Microglia

Pericytes

SMC

Next steps for further analysis

  • Level 2 analysis
    • Comparisons of cell types across fractions and disease state (numbers, expression, etc)
    • Genotyping
    • integrating published data (e.g. Yang et al)

A photo of a sign post pointing in several directions

Next project

  • BHF grant - building a vascular atlas
  • 5 regions from 60 post-mortem brains
    • 20 with severe SVD, 20 none/mil/moderate SVD and 20 sudden death controls
    • Same 2 vascular/parenchymal fractions = 600 samples for sequencing
  • Subset of samples for ATACseq and spatial transcriptomics
  • Again using scFlow/ADDI for standardised analysis/data access

Thanks for listening

References

Gabriel Mateus Bernardo Harrington - Source code on GitHub - GNU GPLv3 license.

Pendlebury, Sarah T., and Peter M. Rothwell. 2009. “Prevalence, Incidence, and Factors Associated with Pre-Stroke and Post-Stroke Dementia: A Systematic Review and Meta-Analysis.” The Lancet Neurology 8 (11): 1006–18. https://doi.org/10.1016/S1474-4422(09)70236-4.
Schaeffer, Samantha, and Costantino Iadecola. 2021. “Revisiting the Neurovascular Unit.” Nature Neuroscience 24 (9): 1198–1209. https://doi.org/10.1038/s41593-021-00904-7.
Warren, Katherine Elizabeth. 2018. “Beyond the Blood:Brain Barrier: The Importance of Central Nervous System (CNS) Pharmacokinetics for the Treatment of CNS Tumors, Including Diffuse Intrinsic Pontine Glioma.” Frontiers in Oncology 8.
Yang, Andrew C., Ryan T. Vest, Fabian Kern, Davis P. Lee, Maayan Agam, Christina A. Maat, Patricia M. Losada, et al. 2022. “A Human Brain Vascular Atlas Reveals Diverse Mediators of Alzheimer’s Risk.” Nature 603 (7903): 885–92. https://doi.org/10.1038/s41586-021-04369-3.